Method for detecting biological activities in a specimen

ABSTRACT

A method, a sensor and apparatus for detecting biological activities in a specimen, for example in a blood sample, are provided in which a sealable container is sealed with a culture medium therein into which the sample is introduced, metabolic processes are enhanced in the presence of microorganisms in the sample and changes taking place in the concentrations of the substances. Such processes are detected and monitored with an excitation and detection assembly assigned to concentration sensors, in the form of optodes which are optically coupled to the excitation and detection assembly and to thereby to an evaluation unit for determining concentration changes of the substances over time as indications of the presence of microorganisms.

This is a division of application Ser. No. 07/501,123 filed Mar. 29,1990, issued as U.S. Pat. No. 5,266,486, which is in turn acontinuation-in-part of Ser. No. 474,786 filed Mar. 29, 1990, issued asU.S. Pat. No. 5,217,875.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method and to an apparatus fordetecting biological activities in a specimen where the specimen and aculture medium are introduced into a sealable container and are exposedto conditions enabling metabolic processes to take place in the presenceof microorganisms in the sample, the concentration of the initialsubstances being lowered and that of metabolic products being raised.

2. Description of the Prior Art

In many applications it is necessary to determine quickly whether aspecimen is contaminated by microorganisms, such as bacteria, inparticular in medical applications, in the pharmaceutical industry, foodindustry, or in environmental protection activities. The term "specimen"has a most comprehensive meaning here, including substances such assolid and liquid biological material (e.g. blood), food samples, such asfrozen foods and preserves or canned foods, packaging material, clinicalinstruments and laboratory equipment, or samples taken from theirsurfaces, medical apparatus, first-aid and dressing material, soil andwater samples, particularly samples of drinking water.

For a long time purely manual methods have been used in which thespecimen to be assessed is placed in a culture bottle containing aliquid culture medium, and the growth of the culture is inspected onlyvisually at given time intervals, and the type of presence of amicroorganism is inferred from this observation by subculturing theliquid culture medium to a solid culture medium.

In addition, some technical procedures and devices are known, with whichthe biological activities in a sample are caused by microorganisms maybe determined, and where the CO₂ produced by the metabolism of themicroorganism, or rather, the change in CO₂ content, is employed as ameasurement for determining the biological activity.

It is a known procedure, for example, to bottle the sample to beassessed together with a radioactively-labeled liquid culture medium andto test the atmosphere over the culture medium for radioactive gases,following which the presence of microorganisms in the sample may bedetermined.

Measuring systems of this type are described in U.S. Pat. Nos. 3,076,679and 3,935,073, for example, fully incorporated herein by this reference.Although such systems are quick and reliable, they have certaindisadvantages, i.e. radioactive substances must be handled and samplesmust be repeatedly taken from the gas space above the culture medium forfrequent monitoring. When the samples are removed from the gas space,the remaining samples to be monitored may easily be contaminated by thesample-taking element and measuring errors may occur.

In the European application 0 158 497, a system is disclosed in whichthe biological activity of the specimen is determined by means ofinfrared absorption. In this method, a specimen is introduced into asealable vessel containing a liquid culture medium, and is tested forthe presence of microorganisms. The vessel is subjected to specificconditions, i.e. certain temperatures are maintained over given periodsof time, thus enhancing the metabolism of the microorganisms, duringwhich process CO₂ is produced in the gas space above the culture mediumby conversion of the carbon source. A sample is taken from the gas spaceand introduced into a measuring cell, and the CO₂ content is measured byinfrared absorption. Again, the subsequent samples may be contaminated,and another drawback is that infrared absorption is a less-sensitivemeans of measuring than radioactive labeling.

In order to avoid the problem of cross-contamination, the Europeanapplication 0 104 463 proposes a method and a device which are alsobased on teh detection of CO₂ (produced by metabolic processes) by meansof infrared absorption. In this method, no sample is taken, but infraredradiation is directly transmitted through the wall of the vessel intothe gas space above the culture medium, and its absorption isdetermined. Due to this non-invasive measuring method,cross-contaminations are largely eliminated; the disadvantage of thismethod, however, is its lack of sensitivity compared to radiometricmethods; as well as the fact that the measurement is distorted by othergas components absorbing radiation in the same frequency band as CO₂. Asuitable example is the absorption bands of water vapor. The samplevessels employed must be transparent within a relatively narrowfrequency range, which will only permit the use of specific materialsfor these vessels. An additional disadvantage is that the generation andfiltering of the required infrared radiation is comparatively complexand expensive.

The European application 0 333 253 describes a device and apparatus formonitoring changes in pH and CO₂ in a bacterial culture utilizingoptical absorption measurements of a pH indicator in a matrix. Althoughit is not as sensitive as the radiometric method, it does offer theadvantage of being noninvasive and can be continuously monitored. Theprimary disadvantage is that since color changes are being measured,different optical systems must then be used when the indicator medium ischanged, thereby limiting the apparatus to one or two sensors.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a methodand an apparatus for detecting biological activities in a specimen,which have at least the same sensitivity as radiometric methods, andwhich offer the user a versatile, simple and inexpensive technique,providing him with information on the presence of microorganisms, whileeliminating the danger of cross-contamination.

According to the present invention, the above object is achieved bycontinuously measuring the concentration of at least one (produced orconsumed) substance subject to conversion by metabolic processes,generating a test signal by way of optodes in direct contact with thesubstances to be assessed, and monitoring the changes over time of thetest signal or signals to serve as an indicator for the presence ofmicroorganisms. The optical sensors or optodes which rely on theprinciple of fluorescence attenuation or enhancement, which may beinexpensively mass produced and which are in direct contact with thesubstance to be assessed and attached to a transparent surface of thecontainer (internal filtered effect), permit continuous monitoring inclosed systems, where new optodes are used for each sample, such thatcross-contaminations are eliminated in the most simple variation, forexample, a single sensor may suffice to determine the presence ofmicroorganisms by way of the changes in substance concentrationsrelative to the initial concentrations.

With respect to the aforementioned disadvantage of measuring colorchanges and the requirement for different optical systems when anindicator medium is changed, another object of the present invention isto provide a sensor which allows the monitoring of many optical changeswhile utilizing the same optical system.

According to the present invention, the immediately-foregoing object isachieved by utilizing a inert fluorophore in the optode and measuringthe modulation of the fluorescent output caused by optical changes inthe indicator medium. It also provides for very sensitive continuouslymonitoring an noninvasive system.

The present invention provides that concentrations be measured of atleast one substance from the group of CO₂, O₂, H₊ (pH), NH₄ ⁺, H₂ S, H₂,and metal ions, the indicator medium of the optodes responding to achange in substance concentrations by changing its opticalcharacteristics such that a change in fluorescence intensity from aninert fluorescing component of the sensor is measured.

In particular, the invention provides, for example, for the testing ofblood samples, that the CO₂ concentration be measured continuously andthe detection of microorganisms be determined upon a rise of the CO₂concentration. The invention further provides for using any combinationof sensors to identify the microorganisms completely or partially.

In accordance with another variation, the invention will also permitcontinuous measuring of the O₂ concentration and determination of thechanges in H₂ S , NH₄ and pH. The advantage of this variation is thatthe significant changes in the O₂, NH₄, H₂ S, and pH concentration maybe detected earlier than the detection of changes of CO₂ alone.

The invention also provides that a culture medium containing a carboncompound be introduced into a sealable container, and that the sensor beadded to the culture medium, which responds to the changes in metabolicsubstances content by a change in its fluorescence behavior, and that ablood sample be introduced into the container, which may be subject tometabolic processes in the presence of microorganisms, during whichmetabolic processes occur, and that the content of the container beexposed to excitation radiation and the radiation emitted by thefluorescent component of the sensor be measured, a change influorescence behavior indicating the presence of microorganisms. Forexample, indicator capsules as disclosed in the German application 23 6384 may be added, or rather, microcapsules containing sensors, whosewalls are made from polymerized hydrophilic monomers.

According to the invention, a device is provided for detectingbiological activities in a specimen, comprising a sealable containercontaining a culture medium into which the sample is introduced, andfurther comprising means enabling metabolic processes to take place inthe presence of microorganisms in the sample, and is characterized bythe use of several optodes for simultaneous assessment of severalsubstances whose concentrations are subject to changes by the metabolicprocesses, and by assigning an excitation and detection assembly to eachoptode, which, in turn, is connected with an evaluation unit fordetermining the change over time of the substance concentrations.Combining two optodes (e.g. O₂ and pH) to form a bisensor, or threeoptodes (e.g. CO₂, O₂, pH) to form a trisensor may be of advantage.

At this point, a description shall be provided with respect to theconcepts, types of structure on a system and component basis,technology, principles of the invention and model systems for particularmaterials, all included in what is referred to as Fluorescence IntensityModulation Sensor Technology (FIMST).

The present invention is based on certain fundamental concepts includingthe desire to use an inert fluorescent material in combination with anaqueous emulsion of an indicator to provide significant benefits due to:

1. Selection of a high quantum efficiency fluorophore;

2. Selection of fluorophore that has an absorptions match to aninexpensive and powerful excitation source;

3. Selection of an indicator that is most sensitive to the region ofinterest;

4. Selection of an indicator with a strong absorptions at either, orboth, the excitation or emission wavelength of the fluorophore; and

5. The potential to use the same optical system (source and detector)for sensors detecting different analytes.

The structure of embodiments of the invention may utilize variouscomponents and variations including:

1. Membrane (e.g. gas permeable silicone)

a. single membrane containing both the fluorescent material and theindicator, or

b. a multi-layer structure with the fluorophore and indicator inseparate layers;

2. Fluorescent material, fluorophore

a. contained in the silicone, or

b. contained in an aqueous emulsion;

3. Indicator

a. contained in the aqueous emulsion,

b. contained in the sample solution, or

c. contained in the sensor matrix;

4. Source

a. light emitting diodes (LEDs) including

1. blue 456 nm peak or 470 nm peak,

2. green 560 nm peak, 550--580 nm half intensity,

3. yellow 590 nm peak, 570--610 nm half intensity,

4. high intensity red (red/orange) 645 nm peak, 615--660 nm halfintensity and

5. red 665 nm peak, 650--670 nm half intensity; and

b. other lamps such as tungsten, quartz halogen and neon; and

5. Detector

a. silicon photodiodes,

b. PIN silicon diodes,

c. GaAsP photodiodes, and

d. other photodetectors including photovoltaics, photoresistive devicesand photoconductive devices.

The method disclosed herein has general applicability for thedetermination of any substance for which a colorimetric method and apermeable membrane can be prepared. Various principles may be used inselecting a sensor system and model sensor systems are discussedhereinbelow.

According to the principles of the invention, current optical sensorsusing fluorescence or absorbance-based materials have inherentlimitations. The absorbance and fluorescence-based systems must usedifferent optical systems (light source, filters and detectors) for eachsensor system, depending on the optical change of the indicator medium.This is due to the necessity of providing and measuring differentwavelengths of light for each different optical system.

The disadvantage for direct fluorescence detection of the metabolicsubstances of microorganisms is that the source and detectors for use influorescence are expensive and of limited availability. In addition,direct fluorescence systems have not yet been described for some testsof interest (e.g., hydrogen sulfide and ammonia).

A system constructed in accordance with the present invention has asensor and an optical system that provides for the determination of afluorescent intensity from the sensor. The optical unit has a lightsource that provides light of the appropriate wavelengths to excite thefluorescent material in the sensor. The light source may be filtered toprovide light only at the wavelengths appropriate to the fluorophore. Inthe most simple case, the source is a light-emitting diode that providestransmission only at the wavelengths needed for excitation.

The detector can be any device that will provide an electronic signalproportional to the light intensity. The preferred embodiment uses asilicon photodiode with a filter to select only the fluorescentwavelength. In a preferred variation, the source and detector are heldin a mount that positions the light from the lamp to illuminate thesensor directly above the photodetector. Appropriate optical filters areused to optimize the signal change associated with the optical change inthe indicator medium.

The sensor is composed of two parts, an inert fluorescent material whichprovides the fundamental optical signal and, the indicator material. Inpractical applications, the sensor is placed and attached to atransparent surface inside a sealable container that can be filled withliquid media that supports the growth of microorganisms.

The fluorescent material is selected to provide an optical signalmatched with the source and detector. The quantum efficiency of thefluorescent material will, to an extent, determine the detection rangeof the sensor system. The fluorescent material may be mixed in thesensor with the indicator material, incorporated as a part of the sensormatrix, applied as a coating to the back of the sensor or in any mannerin which the light from the source will pass through the indicatorbefore or after passing through the fluorescent material. The indicatormaterial is selected to provide an optical change in response to theanalyte at the concentrations expected. The sensitivity of the sensorsystem is determined by the amount which the color changes of theindicator interacts with (modulates) the excitation and/or emission ofthe fluorescent signal. In use, the sensor system provides a constantsignal when the concentration of the analyte remains constant. When theanalyte concentration changes, the indicator responds by exhibiting achange in optical properties (e.g., color intensity). This change actsas an optical filter to change the amount of light exciting or emittedfrom the fluorescent substrate. This change in light is detected as anamplitude change (modulation) by the appropriately-filteredphotodetector.

A number of different configurations are possible in this sensor systemincluding having separate layers of silicon membrane for the fluorescentmaterial and the indicator. In a preferred variation, the optode iscovered with a dye-impregnated silicone to maintain optical isolationfor the system. Examples of the construction of the sensor are presentedin Tables 1 and 2. An example of using this optical system for thedetection of carbon dioxide, hydrogen sulfide and pH are presented. Inthese model systems, the optical unit remains the same even though theindicator component changes.

Several model system examples will now be provided.

The optical system may comprise a yellow LED source and an output of 63mcd at a peak wavelength of 590 nm. The detector is a silicon photodiodewith a peak sensitivity at 720 nm and 0.37 W/A sensitivity at 660 nm.

A model system for the detection of CO₂ is disclosed below. Thefluorescent material carboxnaphthofluorescein is suspended in a matrixof carbon dioxide-permeable silicone polymer. Included in this matrix isan aqueous emulsion of Bromthymol Blue indicator. The fluorescentmaterial is excited by using the yellow LED with a peak emission of 590nm and band width of 40 nm. This corresponds well to the peak absorbancefor the fluorescent material carboxynaphthofluorescein at 598 nm. Theemission of the fluorescent material is maximum at 660 nm. The sensor isan equilibrium with the carbon dioxide present in the media and thisresults in the aqueous emulsion of the sensor having a pH of greaterthan 7.6. If bacteria are present in the sample, the CO₂ producedthereby defuses through the silicone membrane into the aqueous emulsionand reduces the pH of the emulsion. As the pH is reduced, more of theBromthymol Blue indicator is converted to the acid form and the color ofthe emulsion changes from blue to yellow (e.g., peak absorbance changesfrom 617 nm to 470 nm and the yellow light is not strongly filteredout).

When the indicator is in the base form (blue) it filters out theexcitation light and no fluorescent signal is detected. As the pH andthe emulsion decreases and more of the base form is converted to theyellow acid form, and more yellow light is allowed to reach thefluorescent material and an optical signal is detected. The lightintensity is found to increase with an increase in CO₂ concentration.Experimentally, however, the operational amplifier inverts the signaland in FIG. 3 the voltage signal is decreased with an increase in carbondioxide.

A model system for the detection of hydrogen sulfide is set forth below.This system acts as a probe, that is, it responds to the presence of H₂S in an irreversible manner. For microorganism detection, the system hasa constant high level of signal in the absence of H₂ S. The detection ofthe presence of H₂ S will be indicative of a particular group ofmicroorganisms.

The system comprises a fluorescent base material,carboxynaphthofluorescein, suspended in a hydrogen sulfide-permeablesilicone polymer. Included in the silicone matrix is a dilute aqueousemulsion of lead acetate. The sensor is illuminated by the yellow LED.The resultant fluorescent signal is detected by the silicone photodiode.In use, the probe provides a constant signal unless there aremicroorganisms in the sample container that produce hydrogen sulfide.The hydrogen sulfide is soluble in the liquid media and will diffuseinto the silicone membrane. Once inside, the hydrogen sulfide willdiffuse into the aqueous emulsion containing the colorless lead acetatesolution. The hydrogen sulfide reacts with the aqueous lead acetate toproduce an insoluble lead sulfide.

The lead sulfide precipitate is black in color and strongly absorbs andscatters both the excitation and emission light. This results in adecrease in the fluorescent signal at the photodiode detector. Thesignal will be proportional to the amount of hydrogen sulfide produceduntil the available lead acetate is completely reacted.

A model sensor system for pH measurement uses the same Bromthymol Blueindicator as does the carbon dioxide sensor. However, instead of havingthe gas-permeable silicon membrane, this sensor has the indicatorimmobilized on a support and protected by a hydrogen ion-permeablemembrane. As the pH of the media changes, the ratio of Bromthymol Blueindicator in the acid to base form changes in a manner proportional tothe hydrogen ion concentration. This change in color provides a changein the fluorescent signal that is also proportional to the hydrogen ionconcentration. This value can be used to determine the pH, as pH=-log(hydrogen ion concentration). The range of this sensor is limited to therange in color change of Bromthymol Blue, 6.2 to 7.6 pH. This range,however, covers the physiological pH range. Should an increase in therange be required, there are other sensor systems listed herein thatcover the range of pH from 4.7 to 8.0.

                  TABLE I                                                         ______________________________________                                                                  Direction of light-                                                 Color-    intensity with CO.sub.2                             Indicators:     Change    increase                                            ______________________________________                                        Blue LED (470 nm)                                                             Fluorophores:                                                                 1-Acetoxypyren 3,6,8-trisulfonic acid                                         DCM 4-dicyanomethylene2methy16(p-dimethylaminostyrol)                         4H-pyran                                                                      p-Nitrophenol   C-Y       increase                                            m-Dinitrobenzoyleneurea                                                                       C-Y       increase                                            Azolitmin       R-B       decrease                                            Bromxylenol Blue                                                                              Y-G-B     decrease                                            Green LED (560 nm)                                                            Fluorophores:                                                                 7-Aminoactinomycin D                                                          Nile Red                                                                      Rhodamine B                                                                   3,6-Dihydroxy xanthone                                                                        C-B       increase                                            Cleves Acid     C-G       increase                                            Propyl Red      R-Y       decrease                                            Neutral Red     R-Y       decrease                                            Bromcresol Purple                                                                             Y-G-P     increase                                            Alizarin        Y-R       increase                                            Yellow LED (590 nm)                                                           Fluorophores:                                                                 Thionin                                                                       3,3-Dimethyloxadicarbocyanine                                                 Carboxynaphtho Fluorescein                                                    Naphtho Fluorescein                                                           Sulforhodamine 101                                                            Cleves Acid     C-G       increase                                            Orcinaurine     C-G       increase                                            p-Nitrophenol   C-Y       increase                                            3,6-Dihydroxy xanthone                                                                        C-B       increase                                            Bromxylenol Blue                                                                              Y-G-B     increase                                            Bromthymol Blue Y-B       increase                                            Red LED (645 nm)                                                              Fluorophores:                                                                 3,3-Diethylthiadicarbocyanine                                                 Nile Blue                                                                     Cleves Acid     C-G       increase                                            Azolitmin       R-B       increase                                            Bromcresol Purple                                                                             Y-G-P     decrease                                            Alizarin        Y-R       decrease                                            Propyl Red      R-Y       increase                                            ______________________________________                                    

                                      TABLE II                                    __________________________________________________________________________    FLUORESCENT DYES AND PIGMENTS                                                                           Absorbance                                                                          Fluorescent                                   Abbr.  Dye                Max. nm                                                                             Max. nm                                       __________________________________________________________________________    HPTS   1-Hydroxypyren3,6,8,-trisulfonic acid                                                            460   515                                           APTS   1Acetoxypyren3,6,8-trisulfonic acid                                                              460   515                                           RHO-123                                                                              Rhodamine 123      505   534                                           RHO-110                                                                              Rhodamine 110      510   535                                           EO     EOSIN              518   550                                           7-AAD  7-Aminoactinomycin D                                                                             523   647                                           SFRHO-G                                                                              Sulforhodamine G   529                                                 RHO-6G Rhodamine 6G Perchlorate                                                                         530   556                                           RHO-6G Rhodamine 6G Perchlorate                                                                         530   590                                           EVA    Evans Blue         550   610                                           NIRE   Nile Red Phenoxazon 9                                                                            551   636                                           RHO-B  Rhodamine B        552   580                                           RHO-B  Rhodamine B        554   627                                           PYRO   Pyronin B          555   599                                           SFRHO-B                                                                              Sulforhodamine B   556   575                                           DODC   3,3-Dimethyloxadicarbocyanine                                                                    582   660                                           SFRHO-101                                                                            Sulforhodamine 101 586   607                                           NAPH-FLU                                                                             Naphtho Fluorescein                                                                              594   663                                           CARB-FLU                                                                             Carboxynaphtho Fluorescein                                                                       598   660                                           TION   Thionin            599   850                                           NIBL   Nile Blue A Perchlorate                                                                          628   690                                           DTDC   3,3-Diethylthiadicarbocyanine                                                                    653   760                                           DOTCI  Methyl-DOTCI*      682   718                                           IR     IR 144**           750   848                                           __________________________________________________________________________     *Methyl 3,3'Diethyloxatricarbocyanine iodide                                  **22-(3-Dihydro-1,1-dimethyl-3-(3-sulfopropyl)-2H-benz(e)indol-2-ylidene)    thylidene)-2-2(4-(ethoxycarbonyl)-1piperazinyl)-1-cyclopenten-1-yl)-1,1-di    ethyl-3-(3-sulfopropyl)-1H-benz(e)-indolium Hydroxide inner salt, compound     with N,NDiethylethanamine (1,1).                                         

                  TABLE III                                                       ______________________________________                                                               ACID    COLOR   BASE                                   ABBR.    INDICATORS    FORM    CHANGE  FORM                                   ______________________________________                                        PYR      Propyl Red    4.7     R-Y     6.6                                    NIPH     p-Nitrophenol 4.7     C-Y     7.9                                    ACOL     Azolitmin     5.0     R-B     8.0                                    BROMCRE  Bromcresol Purple                                                                           5.2     Y-G-P   6.8                                    CHLORO   Chlorophenol Red                                                                            5.4     Y-R     6.8                                    DX       3,6-Dihydroxy 5.4     C-B     7.6                                             xanthone                                                             ALI      Alizarin      5.6     Y-R     7.2                                    BROXBL   Bromxylenol Blue                                                                            5.7     Y-G-B   7.5                                    DPD      3,6-Dihydroxy 5.8     B-G     8.2                                             phthalic dini                                                        NITEU    m-Dinitro-    6.0     C-Y     7.8                                             benzoyleneurea                                                       BROMBL   Bromthymol Blue                                                                             6.2     Y-B     7.6                                    AU       Aurin (Aosolic acid)                                                                        6.3     Y-P     6.9                                    PHENRE   Phenol Red    6.4     Y-O-R   8.0                                    CLEV     Cleves Acid   6.5     C-G     7.5                                    ORC      Orcinaurine   6.5     C-G     8.0                                    RES      Resolic acid  6.8     Y-R     8.0                                    NEURE    Neutral Red   6.8     R-Y     8.0                                    ______________________________________                                        pH-Indicators                                                                 B--blue  -500 nm                                                              G--green 510-590 nm                                                           Y--yellow                                                                              590-620 nm                                                           O--orange                                                                              620-640 nm                                                           R--red   640-700 nm                                                           P--purple                                                                              700-750 nm                                                           C--colorless                                                              

As indicated above, according to invention, optodes are provided forselecting detection of at least one substance from the group of Co₂, O₂,A₂ 2, A2, H⁺ (pH), Nh₄ ⁺ metal ions and H₂ S, that are present duringthe metabolic process, as initial, intermediate or the final products.

The excitation and detection assembly may comprise a LED light sourceand a photodiode detector as well as a two-armed optical waveguidetransmitting excitation radiation to the optode or optodes at carryingof the optical signal to the detector.

In a preferred variation of the invention, the optodes are combined toform a multilayer sensor.

In a simple structure of the invention, the optodes are attached to theinside of the wall of a transparent container and are connected to theevaluation unit via the excitation and detection assembly that may beplaced flush against the outside of the wall of the container. Theoptodes, which may be mass-produced inexpensively, are attached directlyor with an adhesive to the inner surface of the wall of the samplecontainer, which is then filled with the culture medium, sealed andstored. After the addition of the specimen, for example, a blood sample,the container is thermostat-controlled for the time required for thegrowth of the culture, and is shaken if necessary, whereupon theconcentration of the substance subject to chemical reaction by themetabolic processes is measured, for example, with a suitableoptically-filtered photodiode in close proximity to the optode.

According to the invention, it is possible to place at least the optodesin the gas space of the at least partially transparent container abovethe culture medium mixed with the sample, and to use these optodes formeasuring the concentration of at least one gaseous metabolite.

It is provided in accordance with the further variation of the inventionthat the optodes be located on a transparent stopper used for sealingthe container.

The method will also permit, however, to place the optodes in a portionof the container covered by the culture medium mixed with the sample,possibly at the bottom of the container, and to use the optodes formeasuring the concentration of at least one substance in the culturemedium. With this arrangement, the metabolite is measured immediately atthe place where it is produced, which will permit more rapid assessmentas to whether a culture is positive or negative.

According to a particularly favorable structure of the invention, thereis provided a device for temperature control of the sample, in whichseveral containers are placed in labeled positions at the same time,each container being assigned an excitation and detection assemblytransmitting excitation radiation through the optodes located in eachcontainer and detecting the ensuing optical signal, and the signals ofthe detection assembly are carried to an evaluation unit together with aposition identification signal. The device used for temperature controlmay be configured as a temperature-controlled supporting rack withmultiple optical stations, which will permit a large number of culturemodels, e.g., up to 600, to be monitored simultaneously. As compared toconventional equipment of this kind, no further handling of the samplesis required once they have been filled into their individual bottles,since both the incubating process and the continuous monitoring arefully automated in the device of the present invention. Unlikeconventional measuring techniques, in which the individual culturebottles must be inserted into an evaluation unit by hand once or twicedaily, the technique of taking measurements continuously will allow thepoint in time when a culture becomes positive to be determined withoutdelay. One or more substances involved in the metabolic process may beassessed optically and the presence of microorganisms may be determinedvery quickly. Therefore, a highly-sensitive automatic measuring systempermitting noninvasive, continuous measuring techniques is provided bythe invention. The evaluation unit either includes amicrocomputer/microcontroller indicating the status of each individualcontainer, or it is connected to a computer via an interface.Information useful for bacterial identification would be available withmultiple optodes.

In another structure of the invention, provisions are made for a devicefor temperature control of the sample, which holds several containers atthe same time, and for a feed mechanism or sample changer automaticallytaking the individual containers to a measuring station, in which theoptodes located in each container enter into optical contact with theexcitation and detection assembly. Whereas the variation described inthe foregoing paragraph has no moveable parts at all, this structure hasa conventional sample changer for automatically taking each sample to ameasuring station. The advantage of this arrangement is that theelectronic or electro-optical equipment need not be so elaborate.

According to the invention, it is also possible to fasten the optodes atthe tip of the probe to be inserted into the container, which probecontains light waveguide elements from the excitation and detectionassembly. The probe may be inserted through an opening sealed by aseptum and introduced into the culture medium mixed with the sample orinto the gas space thereabove.

A further variation of the invention provides that the container besealed by a septum which may be punctured with a hollow needle of asampling vessel, the optodes being located in the sampling vessel and aflow connection being established between the gas space of the containerand the optodes via the hollow needle of the sampling vessel, after thesample has been added to the culture medium in the container. As asampling vessel an evacuated vessel may be used, for example, to whoseinner wall surface optodes may be affixed. The sample, e.g. blood, issucked into the container by the vacuum applied to the vessel. Theseptum of the container holding the culture medium is pierced with theneedle and the blood is introduced into the culture bottle. In view ofthe hollow needle, metabolic gases are conveyed from the gas space abovethe culture medium to the sensor, where they are measured. The culturebottle and sampling vessel are preferably designed as single-usearticles that are discarded after use.

BRIEF DESCRIPTION OF THE DRAWINGS:

Other objects, features and advantages of the invention, itsorganization, construction and operation will be best understood fromthe following detailed description, taken in conjunction with theaccompanying drawings, on which:

FIG. 1 is a schematic representation of a device for practicing theinvention;

FIGS. 2a, 2b, 2c, 2d, 2e, 3, and 4 are similar views of variations ofthe device of FIG. 1;

FIG. 5 is a schematic representation of a variation for automaticmeasuring of several containers at the same time;

FIGS. 6 and 7 are schematic representations of variations of thestructure of FIG. 5;

FIGS. 8, 9 and 10 are graphic illustrations of curves of measuredconcentrations; and

FIG. 11 is a graphic illustration of measured curves using the sensor ofthe present invention to detect the growth of a variety of bacteriacommonly found in cases of bacteremia.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The device of FIG. 1 for detecting biological activities in a samplecomprises a sealable, optically-transparent container 1 with an optode 3attached to the inner surface 2 of its wall and bonded by a transparentadhesive layer 4.

Instead of a single optode 3 for a substance to be assessed, two or moreoptodes, 3a, 3b, and 3c may be combined into a multilayer sensor, whichwill permit simultaneous detection of the changes in O₂ and CO₂concentrations and in pH, for example. The individual optodes 3a--3c ortheir indicator media may be stacked in layers one above the other, orthey may be imbedded in a polymer membrane in homogenous distribution.The combination of a C₂ and an O₂ optode into a sensor is described inthe European application O 105 870, for example.

Instead of the optode 3 in the variations discussed below, optodes maybe provided for measuring O2, Co₂, H⁺ (pH), Nh₄ +, H₂ S" and H₂, orrather, a specific combination of these optodes in accordance with theparticular requirements.

The container 1 contains the culture medium 4 with one carbon compound(glucose), for example, which is converted by metabolic processes ofmicroorganisms in the sample and to metabolic product, for example CO₂,during which processes O₂ is being consumed and the pH is subject tochange. As a consequence, there are changes in the concentration of themetabolic product and the initial substances and the gas space 6 abovethe culture medium 5 and in the culture medium itself, which aredetected by way of the optodes 3a, 3b, and 3c placed at the bottom 7 ofthe container 1 in FIG. 1. The excitation and detection assembly 8comprises a light source 9, a detector 11 and a two-armed lightwaveguide 10, one of whose arms is coupled to the light source 9 and theother of which is coupled to the detector 11. The end 12 of the lightwaveguide is placed flush against the exterior 13 of the wall of thecontainer, transmitting excitation radiation towards the optodes 3a, 3band 3c through the transparent wall of the container, while receivingthe optical signal, e.g., the fluorescence radiation emitted by theoptodes.

The use of a suitable filter 31, for example, a filter disc, at front ofthe detector 11 will ensure that the signals are assigned to theircorresponding optodes 3a, 3b, 3c.

By way of a line 14, the detector signals are transmitted to anevaluation unit 15 in which the change over time, e.g., of the CO₂content is determined and the status of the sample is indicated via adisplay 16.

The conditions in the container necessary for the metabolic processesare maintained by way of the unit 17, which is mainly responsible forproper temperature control of the sample, and is connected with theevaluation unit 15 via a control lead 18.

Instead of a heating unit 17, an air heating element may be used forsample temperature control of the variation of FIG. 1 and all subsequentvariations.

The structure illustrated in FIG. 2a differs from that of FIG. 1 only bythe fact that the optode 3 is located in the gas space 6 of thecontainer 1 and that the metabolites may be measured only. In thisinstance, temperature control is performed via the bottom 7 of thecontainer 1. In a structure according to FIG. 2b, the optode 3, or theoptodes 3a, 3b, may be attached to a stopper 1' sealing the container 1.The light waveguide 10 may go either through this stopper 1' or it maybe placed on the exterior of the transparent stopper 1', as is shown inFIG. 2b. FIGS. 2c, 2d and 2e illustrate other modifications of FIG. 1which utilize LEDs and photodiode detectors even though each optode hasa different chemistry and produces a different optical change (e.g.,color). The sensor's optical system is being used because thefluorescence is being measured.

In another structure as presented in FIG. 3, the optode 3 is attached tothe tip of a probe 19 receiving the end of the two-armed light waveguide10. The probe 19 is introduced into the container 1 through the opening21 sealed by a septum 20, and may be axially shifted along in thedirection of the arrow 22, permitting measurements to be taken both inthe gas space 6 and in the culture medium 5.

In the structure illustrated in FIG. 4, the optode 3, which is used, forexample, for measuring O₂ and CO₂, is not located in the container 1,but is placed in a sampling vessel 23. In order to introduce the sampleinto the culture bottle, the septum 20 of the container 1 is puncturedwith the hollow needle 24 of the sampling vessel 23, permitting thesample to enter the culture medium. Via the hollow needle 24 a gasexchange will take place between the gas space 6 and the interior 25 ofthe sampling vessel 23 such that the change in the concentration of CO₂and O₂ may be determined with the use of the excitation and detectionassembly 8 which is symbolically indicated by the light waveguide 10.

A particularly favorable structure is illustrated in FIG. 5 comprising adevice 26 configured as a temperature-controlled supporting rack, whichwill hold several containers 1 at the same time. The containers areinserted in labeled positions and are arranged in several rows,permitting temperature control and continuous monitoring of up to 600containers simultaneously. Each container is assigned a two-armed lightwaveguide 10 located in the supporting rack 26 and providing the optodes3a--3c at the bottom of each container 1 with excitation radiation. Thecorresponding optical signals are delivered to the individual detectors11 connected to the evaluation unit 15 via lines 14. The individualreadings delivered to the evaluation unit 15 are accompanied by suitableposition identification signals such that the individual values may bedirectly assigned to the corresponding sample.

A structure as presented in FIG. 6 provides that each individualcontainer 1 be connected with a LED 27 located in the supporting rack 20and with a photodiode 28, possibly in conjunction with photo elements.In this manner, a most compact device is obtained which is characterizedby the total absence of moveable parts.

FIG. 7 illustrates a structure according to FIG. 6, which contains twooptodes, 3a and 3b, combined into a sensor (e.g., a bisensor), forsimultaneous measurement of O₂ concentration and pH. The optodes areexcited via different LEDs 27 and 27' whose emission radiation isreceived by a common photodiode 28. The corresponding electrical leads29, 29', 30 lead to the evaluation unit not shown here. By means ofknown optical or electronic equipment, the signals of the two optodesmay be separated. Other variations with only one LED for excitation andseveral photodiodes for signal detection are within the scope of thisinvention.

In the graphic illustrations of FIGS. 8 and 9, the time t, or rather,the individual points in time T₀ to T₈ are plotted on the abscissa,while the pH value, the concentration K (or the partial pressure of O₂and CO₂) and the number of bacteria or organisms per unit volume(logarithmic scale) are plotted on the ordinate.

FIG. 8 illustrates the change over time of the parameters O₂, CO₂ and pHusing a sample containing Staphylococcus areus. Between the times T₅ andT₆ the concentration of CO₂ is characterized by a significant increaseindicating a positive sample, and about the same time the O₂ and pHlevels decrease.

As opposed to FIG. 8, the pH value in FIG. 9 remains largely unchanged,whereas the O₂ decrease occurs significantly sooner than the CO₂increase. In this instance, a sample with Psaudomonas aeruginosa wasused.

The values provided in FIG. 10 are taken from a sample containingenterobacteria (E. coli). All three parameters change and once again theO₂ decrease was noticed prior to the changing of the other parameters.

The method and device described herein are well-suited for detectingbiological activities and samples, for example, of microorganisms inblood, (bacteriemia, septicemia or pyemia).

The continuous, noninvasive monitoring of specimens helps to obtainfully automated incubation and measuring processes for a large number ofsamples. Positive cultures are quickly identified and erroneous negativefindings are avoided.

Although we have described our invention by reference to particularillustrative embodiments and examples thereof, many changes andmodifications of the invention may become apparent to those skilled inthe art without departing from the spirit and scope of the invention. Wetherefore intend to include within the patent warranted hereon all suchchanges and modifications as may reasonably and properly be includedwithin the scope of our contribution to the art.

We claim:
 1. A method for detecting biological activity in a specimen ina mixture with a culture medium capable of sustaining the growth ofmicroorganisms exhibiting a metabolic activity which alters theconcentration of at least one substance concentration in said mixture,said method comprising the steps of:disposing said mixture in asealable, transparent container having an inner surface and an outersurface; exposing said mixture in said container to light from a lightsource disposed externally of said container; attaching a sensor to saidinner surface of said container having a sensor surface exposed to saidmixture and permeable by said substance, said sensor including an inertfluorophore responsive to light from said light source to emitfluorescent radiation of a predetermined wavelength; detecting saidfluorescent radiation; and dispersing an indicator means in saidcontainer, between said fluorophore and said light source, which isoptically responsive to a change in the concentration of said substanceand thereby amplitude-modulating said fluorescent radiation of saidpredetermined wavelength to a degree corresponding to said change insaid concentration of said substance.
 2. A method as claimed in claim 1wherein the step of dispersing an indicator means in said container isfurther defined by dispersing an indicator means in said containerbetween said fluorophore and said light source for altering light fromsaid light source, before said light reaches said inert fluorophore toamplitude-modulate said radiation emitted by said inert fluorophore. 3.A method as claimed in claim 1 wherein the step of dispersing anindicator means in said container is further defined by dispersing anindicator means in said container between said fluorophore and saidlight source for altering said fluorescent radiation emitted by saidinert fluorophore, to amplitude-modulate said radiation emitted by saidinert fluorophore.
 4. A method as claimed in claim 1 comprising theadditional steps of:selecting said inert fluorophore from the groupconsisting of Thionin, 3,3-Dimethyloxadicarbocyanine, CarboxynapthoFluorescein, Naptho Fluorescein and Sulforhodamine 101; and selectingsaid indicator means from the group consisting of Cleves Acid,Orcinaurine, p-Nitrophenol, 3,6-Dihydroxy xanthone, Bromxylenol Blue andBromthymol Blue.
 5. A method as claimed in claim 1 comprising theadditional steps of:containing said inert fluorophore in a CO₂-permeable silicone polymer matrix material of said sensor; selectingsaid inert fluorophore from the group consisting of3,3-Diethylthiadicarbocyanine and Nile Blue; and selecting saidindicator means from the group consisting of Cleves Acid, Azolitmin,Bromcresol Purple, Alizarin and Propyl Red.
 6. A method as claimed inclaim 1 comprising the additional steps of:selecting said inertfluorophore from the group of dyes and pigments consisting of1-Hydroxypyren 3,6,8-trisulfonic acid, 1-Acetoxypyren 3,6,8-trisulfonicacid, Rhodamine 123, Rhodamine 110, EOSIN, 7-Aminoactinomycin D,Sulforhodamine G, Rhodamine 6 G Perchlorate (530 nm absorbance, 556 nmfluorescence), Rhodamine 6G Perchlorate (530 nm absorbance, 590 nmfluorescence), Nile Red Phenoxazon 9, Rhodamine B (552 nm absorbance,580 nm fluorescence), Rhodamine B (554 nm absorbance, 627 nmfluorescence), Pyronin B, Sulforhodamine B,3,3-Dimethyloxadicarbocyanine, Sulforhodamine 101, Naptho Fluorescein,Carboxynaptho Fluorescein, Thionin, Nile Blue A Perchlorate,3,3-Diethylthiadicarbocyanine, methyl 3,3' diethyloxatricarbocyamineiodide and 2-2-(3-Dihydro-1,1-dimethyl-3-(3-sulfopropyl)-2H-benz(e)indol-2-ylidene)ethylidene)-2-2(4-(ethoxycarbonyl)-1piperanzinyl)-1-cyclopenten-1-yl)-1,1-dimethyl-3-(3-sulfopropyl)-1H-benz(e)-indoliumHydroxide inner salt, compound with N,N-Diethylethanamine (1,1); andselecting said indicator means from the group consisting of Propyl Red,p-Nitrophenol, Azolitmin, Bromcresol Purple, Chlorophenol Red, 3,6Dihydroxy xanthone, Alizarin, Bromxylenol Blue, 3,6 Dihydroxy phthalicdini, m-Dintrobenzoyleneurea, Bromthymol Blue, Aurin, Phenol Red, ClevesAcid, Orcinaurine, Resolic acid, and Natural Red.